Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium.
ABSTRACT Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO3- secretory pathway that was defective in CF: (i). ASL [HCO3-] was higher in NL than CF ASL; (ii). activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii). NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent HCO3- secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.
- SourceAvailable from: Leah Reznikov[Show abstract] [Hide abstract]
ABSTRACT: The pulmonary airways are continuously exposed to bacteria. As a first line of defense against infection, the airway surface liquid (ASL) contains a complex mixture of antimicrobial factors that kill inhaled and aspirated bacteria. The composition of ASL is critical for antimicrobial effectiveness. For example, in cystic fibrosis an abnormally acidic ASL inhibits antimicrobial activity. Here, we tested the effect of pH on the activity of an ASL defensin, human β-defensin-3 (hBD-3), and the cathelicidin-related peptide, LL-37. We found that reducing pH from 8.0 to 6.8 reduced the ability of both peptides to kill Staphylococcus aureus. An acidic pH also attenuated LL-37 killing of Pseudomonas aeruginosa. In addition, we discovered synergism between hBD-3 and LL-37 in killing S. aureus. LL-37 and lysozyme were also synergistic. Importantly, an acidic pH reduced the synergistic effects of combinations of ASL antibacterials. These results indicate that an acidic pH reduces the activity of individual ASL antimicrobials, impairs synergism between them, and thus may disrupt an important airway host defense mechanism.Proceedings of the National Academy of Sciences 11/2014; · 9.81 Impact Factor
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ABSTRACT: The localization of Burkholderia cepacia complex (Bcc) bacteria in the cystic fibrosis (CF) lung, alone or during co-infection with Pseudomonas (P.) aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa on 21 co-/or singly-infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc was predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus, but not as "biofilm-like structures". In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during co-infection with Bcc. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented under anaerobic conditions with alternative electron acceptors, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by HPLC. Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically, but not aerobically. We propose that Bcc: 1) invades a P. aeruginosa infected CF lung when the airway lumen is anaerobic; 2) inhibits P. aeruginosa biofilm-like growth; and 3) expands the host bacterial niche from mucus to also include macrophages.Infection and Immunity 08/2014; · 4.16 Impact Factor
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ABSTRACT: Cystic Fibrosis (CF) is the most common fatal monogenic disease among Caucasians. While CF affects multiple organ systems, the principle morbidity arises from progressive destruction of lung architecture due to chronic bacterial infection and inflammation. It is characterized by an innate immune defect that results in colonization of the airways with bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa from an early age. Within the airway microenvironment the innate immune cells including epithelial cells, neutrophils, and macrophages have all been implicated in the host defense defect. The neutrophil, however, is the principal effector cell facilitating bacterial killing, but also participates in lung damage. This is evidenced by a disproportionately elevated neutrophil burden in the airways and increased neutrophil products capable of tissue degradation, such as neutrophil elastase. The CF airways also contain an abundance of nuclear material that may be originating from neutrophils. Neutrophil extracellular traps (NETs) are the product of a novel neutrophil death process that involves the expulsion of nuclear material embedded with histones, proteases, and antimicrobial proteins and peptides. NETs have been postulated to contribute to the bacterial killing capacity of neutrophils, however they also function as a source of proteases and other neutrophil products that may contribute to lung injury. Targeting nuclear material with inhaled DNase therapy improves lung function and reduces exacerbations in CF and some of these effects may be due to the degradation of NETs. We critically discuss the evidence for an antimicrobial function of NETs and their potential to cause lung damage and inflammation. We propose that CF animal models that recapitulate the human CF phenotype such as the CFTR−/− pig may be useful in further elucidating a role for NETs.Antibiotics. 01/2015; 4(1):62-75.
Abnormal surface liquid pH regulation by cultured
cystic fibrosis bronchial epithelium
Raymond D. Coakley*, Barbara R. Grubb*, Anthony M. Paradiso*, John T. Gatzy†, Larry G. Johnson*, Sylvia M. Kreda*,
Wanda K. O’Neal*, and Richard C. Boucher*‡
*Cystic Fibrosis?Pulmonary Research and Treatment Center, School of Medicine, and†Department of Pharmacology, University of North Carolina,
Chapel Hill, NC 27599-7248
Edited by Lily Y. Jan, University of California School of Medicine, San Francisco, CA, and approved October 30, 2003 (received for review July 19, 2002)
Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-
participate in airway surface liquid pH regulation and contribute to
lung defense. We measured pH and ionic composition in apical
and after challenge with luminal acid loads. Under basal conditions,
regulatory paths that contributed to basal pH were identified in the
apical membrane of airway epithelia, and their activities were mea-
sured. We detected a ouabain-sensitive (nongastric) H?,K?-ATPase
that acidified ASL, but its activity was not different in NL and CF
cultures. We also detected the following evidence for a CFTR-depen-
was higher in NL than CF ASL; (ii) activating CFTR with forskolin?3-
isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL;
and (iii) NL airway epithelia more rapidly and effectively alkalinized
ASL in response to a luminal acid challenge than CF epithelia. We
conclude that cultured human CF bronchial epithelial pHASLis abnor-
mally regulated under basal conditions because of absent CFTR-
impaired capacity to respond to airway conditions associated with
acidification of ASL.
regulator (CFTR) in the apical membrane of airway epithelial cells
a universally accepted paradigm linking abnormal ion transport to
fatal lung disease in CF is lacking. Abnormal pH regulation of
apical surface liquid (ASL) could contribute to CF pathogenesis,
because biological processes on airway surfaces are pH-sensitive.
Observations of more acidic pancreatic and seminal secretions in
abnormally acidic luminal solutions, reflecting the fact that bicar-
bonate can permeate CFTR (5).
Difficulties sampling human ASL have limited ASL pH mea-
surements in vivo. Studies of cultured human airway epithelial cells
support CFTR-dependent apical bicarbonate conductance (6–8),
but have not demonstrated pHASL dysregulation on CF airway
epithelia. Although in vivo measurements of tracheal surface liquid
in normal and CFTR knockout mice failed to reveal a significant
pulmonary phenotype in the CF knockout mouse (10) and the
paucity of CFTR expression in the murine trachea (11) argue that
these data may have limited relevance to human normal and CF
The inability to speculate on consequences of reduced CFTR-
data on other pH regulatory pathways in airway epithelia; no
evidence for an apical membrane Na??H?exchanger (12, 13) or
anion exchanger has been reported. A luminal H?,K?-ATPase
(exchanging luminal K?for cytosolic protons, and acidifying
pHASL) was postulated in nasal airway epithelia (14), although
its molecular identification and functional role were not tested,
?secretion and that this defect can lead to an
ystic fibrosis (CF) is a fatal hereditary disease caused by lack of
functional expression of the CF transmembrane conductance
?secretion into CF ASL reflects the paucity of
and recent data did not confirm its presence (15). A paracellular
pathway could also contribute to pHASLregulation, but the per-
meability of this pathway to H?or HCO3
We hypothesized that airway pHASLregulation involves apical
membrane ion transport processes mediated by CFTR and by a
H?,K?-ATPase, in parallel with the paracellular movement of H?
in CF airway epithelia leads to an inability to balance proton
secretion and alkalinize ASL. Therefore, we measured the pH and
ionic composition of the ASL of primary cultures of CF and NL
respiratory epithelia under basal conditions and after airway lumi-
nal acid challenge.
?has not been studied.
Cell Culture. Human bronchial epithelial cells were obtained at the
time of lung transplantation from main stem?lobar bronchi of CF
(n ? 4, one primary ciliary dyskinesia, two non-CF bronchiectasis,
one primary pulmonary hypertension) lungs, using protocols ap-
proved by the Institutional Committee on the Protection of the
Rights of Human Subjects. Nasal epithelial cells were obtained
from resected nasal polyps. Disaggregated airway epithelial cells
were harvested, seeded, and cultured on 1.13-cm2Transwell Col
porous filters (pore diameter ? 0.45 ?m, Costar, Cambridge, MA)
as described (16, 17), and studied 10–14 days after becoming
confluent. Function was assessed by measuring transepithelial
bioelectric potential difference (PD). The transepithelial resistance
of CF and NL cultures was similar (CF 510 ? 24 vs. NL 534 ? 36
??cm2). For studies of intracellular pH (pHi) regulation,
airway cells were studied as described (12). Freshly excised bron-
chial tissue specimens were used for H?,K?-ATPase expression
Collection of ASL and Measurement of Ionic Composition. The apical
culture surface was washed (PBS). Test media was added to the
apical compartment, aspirated, and reapplied. After specified time
intervals, aliquots (1–5 ?l) of ASL were removed with a constant
bore microcapillary pipette as described (17). Na?and K?were
measured by flame emission spectroscopy and Cl?was measured
by amperometric titration (18). HCO3
enzymatic reactions and spectrophotometric analysis of NAD.
Lactate was measured by a NAD-coupled assay (Sigma).
?was measured by coupled
Measurement of ASL pH. We developed a novel technique to
measure pH with pH-sensitive microelectrodes (Microelectrodes,
Bedford, NH) in small-volume samples that could be quickly
temperature, water vapor, and gas equilibrated. Microaliquots
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ASL, apical surface liquid; CF, cystic fibrosis; CFTR, CF transmembrane
conductance regulator; NL, normal; PD, potential difference; pHi, intracellular pH; IBMX,
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2003 by The National Academy of Sciences of the USA
December 23, 2003 ?
vol. 100 ?
no. 26 ?
of a section (0.5 cm) of CO2-permeable silicone tubing (Helix
Medical, Malvern, PA; 0.025 in inner diameter, 0.047 in outer
diameter). The pH microelectrode was inserted into the sample by
stretching the end of the tubing containing the sample over the
microelectrode tip, the tight fit trapping a thin layer of liquid
electrodes made contact with the sample. The microelectrode and
tubing were placed in a water bath that was continually gassed and
equilibrated with 5% CO2. A column of air in the tubing, distal to
the electrode, prevented water from reaching the sample. CO2
equilibration was complete within 2 min, as evidenced by a stable
pH. Measurements were accurate and reproducible within ?0.01
Measurement of H?,K?-ATPase Expression. Freshly excised tissue
specimens were rapidly dissected and snap-frozen in OCT embed-
ding compound. Thin sections (6–10 ?m) were cut by a cryotome,
mounted on glass slides, and stored at ?80°C until analysis. Well
differentiated human bronchial cultures and frozen tissue sections
were fixed in 4% paraformaldehyde (room temperature, 4 min),
permeabilized in 100% ethanol (?20°C, 4 min), and blocked with
20% goat serum in 50 mM sodium phosphate, pH 7.4?150 mM
NaCl (3 h), before overnight (4°C) incubation with a monoclonal
antibody against H?,K?-ATPase (Sigma) or control IgG (Jackson
ImmunoResearch). Specimens were washed in PBS and incubated
(1 h, 23°C) with Texas Red-conjugated goat anti-mouse IgG
(Jackson ImmunoResearch) and fluorescein-labeled phalloidin
(Molecular Probes). After washing, cultures were excised from the
plastic supports and mounted on glass by using Vectashield con-
taining 4,6-diamidino-2-phenylindole (DAPI) to label nuclei blue
(Vector Laboratories). Confocal analyses used a Leica TCS 4D
confocal microscope. All of the channels (red, green, and blue)
were registered by independent and sequential scan of the speci-
mens. Cultures were scanned in the xz axis and tissue sections were
scanned in the xy axis.
Nongastric H?,K?-ATPase mRNA Expression. RNA was isolated by
using the Qiagen RNAeasy RNA purification kit with DNase
treatment according to the manufacturer’s instructions (Qiagen,
RNA and random primers according to manufacturer’s instruc-
tions. Control RNA (colon and stomach) was purchased from
Ambion (Austin, TX). PCR was performed by using the Roche
LightCyler Thermocycler and the LightCyler-FastStart DNA Mas-
ter SYBR Green I kit (Roche Diagnostics), using the forward
primer 5?-TCTGAAGAACAACTGCCTG and reverse primer
5?-TACACGTTGTTCAGGGATG. The PCR product corre-
sponds to positions 266–604 of the ATP1AL1 sequence (the
nongastric K?,H?-ATPase, GenBank accession no. U02076).
Primers were designed to span known introns to eliminate signal
from contaminating DNA. Positive control primers for the cyclo-
philin cDNA were 5?-CCGTGTTCTTCGACATTGCC (forward)
and 5?-ACACCACATGCTTGCCATCC (reverse). For PCR, 1 ?l
of cDNA template was used in a 20-?l reaction volume containing
3 mM MgCl2, 500 nM of each primer, and 1? master mix.
Amplification conditions were as follows: 1 cycle at 95°C for 10 min
and 45 cycles of 95°C for 15 sec, 55°C for 5 sec, and 72°C for 18 sec.
The amplified products were separated on a 1.2% agarose gel.
cAMP-Mediated Regulation of pHASL in CF and NL Cultures. After
washing of the apical surfaces of CF and NL bronchial tissues, 100
?l of HCO3
5.6) was added apically. Forskolin (10?5M) and 3-isobutyl-1-
methylxanthine (IBMX) (2 ? 10?4M) or vehicle control were
?- and K?-free saline Ringer’s solution (140 mM
added to the apical and basolateral medium. ASL was sampled for
measurements of pH and ionic composition.
Assessment of Paracellular HCO3
nasal epithelium (n ? 8, triplicate preparations) were mounted
in a modified Ussing chamber. The CF apical membrane ionic
conductance is virtually zero in the presence of luminal amilo-
ride (10?4M), because CFTR is absent and Na?channels are
blocked (19), and basal calcium activated chloride conductance
is minimal in the absence of a signal for increased intracellular
Ca2?(20). Consequently, voltage changes induced by changes in
apical vs. basolateral anion bath concentrations are likely dom-
inated by bi-ionic PDs across the paracellular shunt (21). The
basolateral compartment was continually perfused with KBR
solution (140 mM Na??125 mM Cl??25 mM HCO3
K??1.2 mM Ca2??1.2 mM Mg2??2.5 mM PO4
cose). To test the relative paracellular anion permselectivity of
the shunt, the lumen was perfused with the following glucose-
free solutions: (i) KBR, (ii) 25 mM Cl?and 125 mM HCO3
test relative Cl?vs. HCO3
permeability. The solution’s cationic composition was identical
(140 mM Na?, 5 mM K?, 1.2 mM Ca2?, 1.2 mM Mg2?, 2.5 mM
bridges linked through calomel half-cells to an electrometer.
?Permselectivity. Cultures of CF
3??2.5 mM glu-
?permeability, and (iii) 25 mM Cl?, 25
?, and 100 mM gluconate to test Cl?vs. gluconate
?). Transepithelial PDs were recorded from 3 M KCl agar
Response of NL and CF Epithelia to Acidification of Apical Liquid. To
test the response to luminal acidification, we exposed the luminal
isotonic saline Ringer’s solution (140 mM Na??140 mM Cl?1.2
mM Ca2??1.2 mM Mg2??2.5 mM PO4
with HCl). After washing with PBS and once with the acidified
Ringer’s solution, 100 ?l of acidified Ringer’s solution was added
apically, and sampled for pH measurement. The basolateral bath
was a KBR solution containing 25 mM HCO3
buffer (pH of both was 7.4). pHimeasurements during solution
changes were made with a RatioMaster fluorimeter (Photon Tech-
nology, Brunswick, NJ) attached via fiber optics to a microscope
(Zeiss), using 2?,7?-bis(carboxyethyl)-4 or 5-carboxyfluorescein
(BCECF) fluorescence (22).
3?, pH adjusted to pH 3.0
?or 25 mM Hepes as
When more than two groups were analyzed, a one-way analysis of
variance analysis was performed with a Student–Newman–Keuls
posttest to isolate differences between groups, whereas response of
a variable in different groups over time was tested with two-way
(GraphPad, San Diego).
ASL pH and HCO3
Epithelium. CF and NL cultures acidified ASL over time (Fig. 1A).
However, acidification rates over the first 6 h were greater for CF
and 48 (P ? 0.01) hours. In parallel, ASL HCO3
decreased at a greater rate in CF vs. NL cultures (Fig. 1B).
ASL acidification over a prolonged period could be affected by
acidification of basolateral media. Basolateral pH had acidified at
24 h, likely reflecting substrate depletion and lactate accumulation,
but was similar in CF and NL cultures (7.29 ? 0.02 vs. 7.31 ? 0.03,
P ? 0.8). Because blood flow would be expected to preserve
substrate concentrations and basolateral pH at ?7.4 in vivo, we
tested the effect of replacing the basolateral solution with fresh
media after 24 h. This led to an alkalinization of pHASLthat was
greater in NL vs. CF cultures (? pH units: NL, 0.33 ? 0.02 vs. CF,
0.23 ? 0.04, P ? 0.05), and resulted in larger basal pHASL
differences between CF and NL cultures.
?Concentrations on CF vs. NL Cultured Bronchial
www.pnas.org?cgi?doi?10.1073?pnas.2634339100Coakley et al.
To determine how cation?anion balance was maintained when
(117 ? 4.3 mEq?liter vs. 134 ? 4.5 mEq?liter, P ? 0.001) and NL
(116.1 ? 4.4 vs. 131.6 ? 3.6 mEq?liter, P ? 0.0001) cultures,
balancing falls in ASL [HCO3
mEq?liter, P ? 0.001) and NL (25.7 ? 1.3 vs. 2.6 ? 0.5, mEq?liter,
0.45 ? 35 mEq?liter, P ? 0.0001) and NL (5.12 ? 0.4 vs. 0.42 ? 0.3
mEq?liter, P ? 0.0001) whereas [Na?] remained stable in samples
from CF (135.8 ? 7.8 vs. 132.9 ? 5.9 Eq?liter) and NL (135.8 ? 6.6
at 24 h did not reveal significant anion gaps (see Fig. 7, which is
published as supporting information on the PNAS web site).
?was depleted in ASL, we measured monovalent ion con-
?] in CF (25.8 ? 1.5 vs. 1.28 ? 0.4
?) and cations (Na?and K?) in ASL on CF and NL cultures
Role of H?,K?-ATPase in Regulation of pHASLin CF and NL Bronchial
Epithelial Cultures. We detected no lactate in ASL (not shown).
Hypothesizing that acidification of ASL on CF and NL cultures
reflected the activity of a H?,K?-ATPase, we varied apical K?
concentrations (0, 5, or 20 mM K?) and demonstrated K?-
epithelial acidification rates were greater over 6 h at each K?
concentration than for NL epithelia (Fig. 2C). Two observations
suggested similar H?,K?-ATPase activity in CF and NL cultures.
of K?removal from ASL (Fig. 2D).
Molecular Identification of H?,K?-ATPase in Human Bronchial Epithe-
lium. We localized H?,K?-ATPase protein to apical membranes of
tissue with confocal immunofluorescence microscopy using a
monoclonal antibody detecting both gastric and nongastric forms
(Fig. 3A). H?,K?-ATPase was also detected in CF tissues (not
shown). Because two H?,K?-ATPase isoforms, a gastric and a
nongastric (colonic) form, are described (23, 24), pharmacological
inhibitor studies were initiated with Sch28080 (which inhibits the
to determine which isoform functionally dominated in airway
epithelium. Sch28080 was a gift from James Kaminski (Schering-
Plough). K?-dependent acidification was inhibited by luminal
ouabain, but not Sch28080 (Fig. 3B), as was the rate of K?removal
from surface liquid (control 7.4 ? 0.34 mEq?hr, ouabain 2.1 ? 0.4
mEq?hr, P ? 0.05; Sch28080 7.1 ? 0.4 mEq?hr, P ? 0.85). PD and
these experiments, suggesting that ouabain had not traversed the
epithelium and inhibited the basolateral Na?,K?-ATPase. These
data support a predominant role for the nongastric form of the
H?,K?-ATPase in airway, a conclusion supported by our detection
of mRNA for nongastric H?,K?-ATPase (ATP1AL1) in cultured
NL airway and colonic, but not gastric, epithelial tissues by using
RT-PCR (Fig. 3C).
Although we have previously detected no resting apical mem-
brane H?or K?conductance in human airway epithelium (12, 13,
21), we tested for a possible role of these conductances in pHASL
regulation under conditions when H?,K?-ATPase activity was
minimized, i.e., under 0 mM K?conditions. The K?channel
blocker, barium, did not alter ASL acidification rates over 6 h
(control 0.066 ? 0.003 vs. barium 0.063 ? 0.004 pH units?hr, n ?
4), arguing against a role for an apical K?conductance and
membrane potential. We probed a role for a proton conductive
pathway in the apical membrane for ASL pH regulation by adding
amiloride (100 ?M) to the lumen to hyperpolarize the apical
membrane potential. Again, we detected no change in ASL acid-
ification rates over 6 h with amiloride (control 0.058 ? 0.006 vs.
amiloride 0.061 ? 0.004 pH units?hr, n ? 4).
cAMP. If more rapid acidification and lower [HCO3
were not caused by abnormal H?secretion rates by the H?,K?-
ATPase, it may reflect absence of CFTR-mediated HCO3
tion. We probed the role of CFTR-mediated HCO3
testing the effect of raising intracellular [cAMP] in CF, NL, and
disease control epithelia on the pH of an apical unbuffered
control epithelia rapidly alkalinized this unbuffered solution, con-
sistent with HCO3
linization accompanied treatment with forskolin?IBMX to raise
intracellular cAMP and activate CFTR (Fig. 4 A and B). In CF
cultures, although an identical rapid alkalinization was observed,
presumably reflecting H??HCO3
lular path (see below), a ‘‘paradoxical’’ acidification, rather than
alkalinization of ASL, occurred (see Fig. 4C), reflecting in part the
absence of CFTR-dependent HCO3
significantly higher than in ASL from forskolin?IBMX-treated NL
?Secretion: Basal Role and Response to Raised Cellular
?] of CF ASL
?-free (pH ? 5.8) ASL solution (Fig. 4). NL and disease
?secretion. A greater and more sustained alka-
?movement through the paracel-
epithelium. One hundred microliters of KBR was applied to the apical surface of
intervals and assayed for pH (A) and HCO3
More acidic ASL on cultured primary human CF vs. normal bronchial
?(B).*, P ? 0.001 pHASLCF vs. NL.
to the apical surface of CF and NL bronchial epithelial cultures. Microaliquots of
apical liquid were assayed for pH. NL (A) and CF (B) cultures exhibit KASL
dependent acidification over 6 h. †, P ? 0.0001, 0 mM vs. 20 mM K?;*, P ? 0.01,
0 mM vs. 5 mM K?. (C) Acidification rates measured in CF and 24 NL cultures in
rates of ASL acidification on CF vs. NL cultures at each [K?]. (D) [K?]ASLdepletion
by CF and NL cultures. One hundred microliters of KBR ([K?] 20 mM?liter) was
applied to the apical surface of cultures of CF and NL bronchial epithelia. Mi-
croaliquots of apical liquid were assayed for [K?]. P ? 0.56.
CF and NL cultures exhibit K?-dependent acidification and ASL K?
Coakley et al.
December 23, 2003 ?
vol. 100 ?
no. 26 ?
cultures (CF-124.6 ? 1.70 mM vs. NL-118.7 ? 1.60 mM, P ? 0.05).
The reciprocal relationship between ASL Cl?and HCO3
that NL but not CF cultures secreted HCO3
Paracellular Pathway Conductivity for HCO3
way epithelia exhibit a relatively ‘‘leaky’’ paracellular pathway that
could participate in pHASLregulation. As best known, the total
ionic permeability of this path is similar in NL and CF (21).
Amiloride-treated CF cultures, lack significant apical ionic con-
ductances, and apical membrane resistances are ?10 K? (21).
Consequently, transepithelial PD changes in response to asymmet-
ric bath ion compositions are dominated by paracellular ionic
gradients. When the apical solution was switched from KBR to the
rected transepithelial concentration gradients for Cl?and HCO3
the PD hyperpolarized (?PD ? ?1.4 ? 0.08 mV, P ? 0.05 vs.
bilateral KBR, Fig. 5), consistent with Cl?exceeding HCO3
permeation via the paracellular path. We continued the sequence
substituted Cl?with the relatively impermeant anion gluconate,
gradients across the cultures for Cl?and gluconate but no gradient
?4.02 ? 0.12 mV, P ? 0.05, vs. low Cl?solution) was observed,
suggesting that the anion permeation sequence of the shunt is Cl?
?and Other Anions. Air-
??low Cl?solution, creating equal but oppositely di-
?permeation. A larger hyperpolarization (?PD ?
Comparison of Response of CF and NL Airway Epithelium to Acid
Challenge. We speculated that the response of CF cultures to
luminal acid challenge was abnormal. We investigated the NL and
CF culture capacity to alkalinize a luminal acidic HCO3
K?-free isosmotic solution (pH 3.0) in the presence and absence of
more slowly and incompletely in CF than NL cultures (Fig. 6).
Comparison experiments performed without basolateral HCO3
demonstrated that basolateral HCO3
to ASL alkalinization in response to acid challenge on NL than CF
cultures (compare Fig. 6 A and B).
The acidic luminal solutions induced small and transient in-
creases in equivalent short circuit current and transient falls in
resistance at 5 min (NL 551 ? 28 before vs. 490 ? 22 after ??cm2;
CF 529 ? 36 before vs. 480 ? 28 after ??cm2, n ? 12, P ? 0.05).
However, no bioelectric evidence of epithelial damage was appar-
ent at this or later time points, consistent with previously published
data (25). Cell viability (by vital dye exclusion) was unaltered.
We tested effects of apical acid challenge on cytosolic pH (pHi)
to ascertain whether pHASLdifferences in CF and NL preparations
after acid challenge reflected differential effects on pHiand, thus,
CF (n ? 3) and NL (n ? 4) culture was seen, nor was baseline pHi
affected by substitution of Hepes for HCO3
(basal pHi: CF-HCO3
0.85; basal pHi: CF Hepes 6.960 ? 0.2 vs. NL Hepes 6.970 ? 0.1,
acidification of pHiin response to apical acidic solutions in the
presence or absence of HCO3
vs. NL HCO3
0.2 vs. NL Hepes 6.780 ? 0.1, P ? 0.9). pHirecovery after luminal
acid challenge was similar in both groups under each condition.
Thus, more effective regulation of pHASLin response to luminal
acid load in NL preparations likely reflected CFTR-mediated
?contributed relatively more
?in the basolateral bath
?6.940 ? 0.2, P ?
?6.960 ? 0.2 vs. NL-HCO3
?(nadir pHi: CF HCO3
?6.750 ? 0.3
?6.750 ? 0.2, P ? 0.85, nadir pHi: CF Hepes 6.760 ?
?secretion, not effects on pHi.
Airway surface liquid is a critical component of lung host
defense. Its pH appears to reflect a balance between active
transcellular ion transport and passive paracellular ion move-
ment. Our experiments defined pathways participating in pHASL
freshly excised bronchus (Lower) were analyzed with confocal microscopy. Specimens were stained with a monoclonal antibody recognizing the ?-subunit of the
ATPase, followed by a Texas Red-labeled secondary antibody and fluorescein-labeled phalloidin. Red and green channels were registered by sequential scanning in
the xz axis (cultures; magnification, ?200) and xy axis (bronchial sections; magnification, ?80). H?,K?-ATPase is heavily expressed in the apical membrane of ciliated
bronchial epithelia from three NL subjects as well as in normal colonic, but not gastric, tissue.
www.pnas.org?cgi?doi?10.1073?pnas.2634339100 Coakley et al.
regulation by NL- and CF-cultured bronchial epithelium under
basal and acid-stressed conditions.
Airway epithelial cultures acidified lumenally applied KBR un-
der basal conditions (Fig. 1). The basal acidification (and HCO3
depletion) rate was more rapid in CF cultures (Fig. 1), and pHASL
acidification likely reflected selective lactate accumulation in the
basolateral compartment. When basal media was replaced with
was accentuated. Taken together, these data show that pHASL
regulation under basal conditions is abnormal in CF airway
ASL acidification reflects, at least in part, apical membrane
H?,K?-ATPase activity. CF and NL cultures exhibited K?-
dependent ASL acidification (Fig. 2). Our studies demonstrated
that (i) K?depletion rates in CF and NL cultures were identical
(Fig. 2D); and (ii) differences in acidification rates between CF and
NL cultures were similar in magnitude, regardless of K?ASL(Fig.
2C), suggesting that H?,K?-ATPase activity was similar in CF and
NL cultures and, thus, not the basis for abnormal pHASLon CF
We characterized the molecular and functional activity of the
H?,K?-ATPase in airway epithelia with complementary ap-
proaches. Immunocytochemical studies verified the apical mem-
brane location of H?,K?-ATPase in cultured proximal human
bronchial epithelium and freshly excised superficial bronchial ep-
ithelium. Pharmacological inhibitor studies showed that that the
nongastric (colonic) isoform was dominant functionally in airway
epithelia, consistent with the demonstration that cultured proximal
human bronchial epithelium expressed mRNA for the nongastric
isoform of the H?,K?-ATPase (Fig. 3).
The simplest explanation for basal pHASLdifferences between
CF and NL cultures is that it reflected the lack of HCO3
by CF airway epithelia, because it was not caused by increased
H?,K?-ATPase-mediated H?secretion. The pHASLalkalinization
is consistent with CFTR-dependent HCO3
?secretion (Fig. 4). With
and acidifies ASL on CF bronchial cultures. We tested the effect of cAMP activa-
tion (10?5M forskolin?200 ?M IBMX bilaterally) on pHASLregulation in NL (Fig.
5A), disease control (Fig. 5B), and CF (Fig. 5C) bronchial epithelial cultures. One
applied apically. Microaliquots of apical liquid were sampled and assayed for
pHASL. (A)*, P ? 0.001 NL control vs. NL forskolin?IBMX. (B)*, P ? 0.001 disease
control vs. disease control ? forskolin?IBMX. (C)*, P ? 0.005 CF control vs. CF
Increased intracellular cAMP alkalinizes ASL on NL bronchial cultures
vidual anions. Primary CF epithelial cultures were mounted in Ussing chambers.
Inhibition of ENaC (Amiloride, 10?4M) eliminated significant apical membrane
conductance because these cultures also lack CFTR. Changes in potential differ-
ence, under these circumstances, thus reflect paracellular ion movement. The
the chloride and bicarbonate concentrations were inverted (imposing equal
‘‘basolateral-to-apical’’ chloride and ‘‘apical-to-basolateral’’ bicarbonate gradi-
ents), and subsequently to one where 95 mM gluconate replaced 95 mM bicar-
bonate in the apical solution (i.e., imposing equal basolateral-to-apical chloride
and apical-to-basolateral gluconate gradients). Results are shown as the PD.
apical ‘‘high apical bicarbonate’’ solution vs. KBR.**, P ? 0.05 apical ‘‘high
gluconate’’ vs. ‘‘high bicarbonate’’ and KBR.
The paracellular path conducts HCO3
?and exhibits selectivity for indi-
applied apically to NL and CF bronchial epithelial cultures. The basolateral me-
dium was KBR or similar solution where 25 mM Na?Hepes replaced 25 mM Na?
?movement after ASL acid-challenge is reduced in
?dependence is observed in NL vs. CF cultures.
Coakley et al.
December 23, 2003 ?
vol. 100 ?
no. 26 ?
respect to possible alternative mechanisms of airway epithelial
changer that might be regulated by CFTR on the apical membrane
of airway epithelium (22), supporting the hypothesis that HCO3
secretion is mediated by CFTR itself and, thus, is electrogenic.
effective means to constantly adjust pHASL. For example, with 25
force for HCO3
electrochemical gradient will favor CFTR-mediated apical HCO3
secretion, limiting acidification. In CF, the HCO3
absent, leading to a failure to buffer H?,K?-ATPase-mediated
proton secretion and acidification of ASL. Note that the paradox-
reported in the gastric parietal cell (26). Taken together, these data
indicate that CF cultures lack the capacity to secrete HCO3
match H?,K?-ATPase-mediated proton secretion into ASL under
basal and cAMP-stimulated conditions, resulting in ‘‘hyperacidifi-
cation’’ of ASL.
pHASLregulation in response to luminal acid challenges is likely
important in lung defense. It has been speculated that the first CF
observation that CF bronchial epithelium less effectively alkalin-
ized an acid challenge to ASL is consistent with this (Fig. 6).
The response to acid challenge was complex. Our data suggested
participation of CFTR-dependent cellular as well as paracellular
likely reflecting transcellular and paracellular HCO3
(Fig. 6). Impairment of realkalinization in CF, compared to NL,
cultures was greatest in the presence of basolateral HCO3
tent with impaired cellular (transapical) HCO3
CF epithelia. In the absence of any known mechanism of apical
to alkalinize pHASLin CF may reflect net H?absorption and?or
suggested that the paracellular pathway is permeable to HCO3
?secretion, we failed to detect evidence of an anion ex-
?in ASL, there is little or no electrochemical driving
?secretion (13). However, if ASL [HCO3
?] falls, the
?exit pathway is
?transport in CF epithelium, the residual ability
?secretion via the paracellular pathway (Fig. 6B), a notion
Indeed, if CFTR is the sole path of HCO3
apical membrane of NL airway epithelial cells, the paracellular
pathway may be the only mode of alkalinizing acidified CF ASL.
Our paracellular permselectivity experiments support a small
paracellular permeability, perhaps dependent on the extreme de-
mucus layer may have intrinsic buffering properties that contribute
to the initial increase in pH. However, when we rechallenged the
preparations with a second acid load, we observed a similar pattern
of recovery (unpublished data), suggesting that pH recovery truly
reflected transepithelial ion movement.
Alterations in pHASLmight contribute to CF pathogenesis. An
abnormally low surface pH may adversely affect mucus viscosity by
altering exposure of hydrophobic regions of mucin molecules as
well as changing the electrostatic charge of their carbohydrate side
chains (29). Thus, in the characteristically depleted CF periciliary
liquid layer, low pHASL would ‘‘tighten’’ adhesive interactions
mucins (17), rendering cough clearance of adherent mucus less
effective (30). Failure of CF epithelia to compensate for an
intraluminal acid load may heighten the inflammatory response in
the airway by interfering with bactericidal activity (31, 32), pro-
moting secretion by immune cells of substances harmful to the lung
Pseudomonas aeruginosa (34).
In conclusion, we have shown that in NL airway epithelia the
activity of a H?,K?-ATPase that acidifies ASL is balanced by the
path can serve to buffer pHASLwith respect to plasma, in part via
linization function is lost by virtue of absent CFTR-mediated
basal conditions and limiting the response to acidic challenges to
?translocation across the
?current and may be best explained by a transient increase in
?secretory function of CFTR. In addition, the paracellular
?permeation. In CF, the airway epithelial cellular alka-
?secretory transport, rendering pHASLmore acidic under
We acknowledge the technical assistance of J. Chadburn, M. Kelly, and
L. Brown. This work was supported by National Institutes of Health
Grant P01 HL 42384 and Cystic Fibrosis Foundation Grant R026.
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www.pnas.org?cgi?doi?10.1073?pnas.2634339100 Coakley et al.